Brandon:LabNotes/Project1/2015-9-21
multiplex combinatorial cellular indexing adaptation to THS-seq using nXTv2 sequence detail and protocol
- adapting THS-seq to combinatorial method sequences detail, however will use 3 barcodes since initial transposition only inserts 1 barcode, and barcode will be 6 base pairs to save on transposon length.
- T7R1-Nextera XT V2 TOP was already validated on Update of THS-seq protocol sequences to use Nextera XT V2 sequences, which adatped from Nextera XT V2 sequences detailed Nextera XT V2 sequences]
Original T7R1-Nextera XT V2 TOP (64 bp) 5'- [CATGG][AATTAATACGACTCACTATAGGGAGA]TCGTCGGCAGCGTC[AGATGTGTATAAGAGACAG] -3’ (T7-nXTv2_i5-1-top) \---/ \-/\---------------------/ \--------------------------------/ spcr T7 rec. T7 req. forward read seq. \-----------------/ ME scT7R1-nXTv2_top (74 bp) 5 basepair spacer removed from 5' end barcode (r5) and {TCCACGCGC} added in, was new sequence added in on combinatorial method, TCCACGC[r5]GC originally 5'- [AATTAATACGACTCACTATAGGGAGA]{TCCACGCGC}(XXXXXX)TCGTCGGCAGCGTC[AGATGTGTATAAGAGACAG] -3’ (scT7_r5XXX_i5_top) \-/\---------------------/ \--------------------------------/ T7 rec. T7 req. forward read seq. \-----------------/ ME Nextera XT V2 Bottom (19 bp with /5Phos/) 5'- /5Phos/CTGTCTCTTATACACATCT -3' (P-T7tspn-bot) T7 description: +1 [CATGG][AAT][TAATACGACTCACTATA|G|GGAGA] [end sequence] rec. T7 req --> [front sequence for optimal binding] After Annealing: 5'- AATTAATACGACTCACTATAGGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3’ (scT7_r5XXX_i5_top) 3'- TCTACACATATTCTCTGTC/5Phos/-5' (P-T7tspn-bot) 5'- /5Phos/CTGTCTCTTATACACATCT -3' (P-T7tspn-bot) 3'- GACAGAGAATATGTGTAGACTGCGACGGCTGCT[r5]CGCGCACCTAGAGGGATATCACTCAGCATAATTAA -5' (scT7_r5XXX_i5_top) Fragments created after transposition: 5'- AATTAATACGACTCACTATAGGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3' 3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNNNNNNNNNN GACAGAGAATATGTGTAGACTGCGACGGCTGCT[r5]CGCGCACCTAGAGGGATATCACTCAGCATAATTAA -5' Upon single insertion, T7 still present in either direction of insertion, IVT off top strand, and bottom: 5'- NNNN--------- /5Phos/CTGTCTCTTATACACATCT -3' 5'- AATTAATACGACTCACTATAGGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN -3’ 3'- NNNNNNNNNNNNN GACAGAGAATATGTGTAGACTGCGACGGCTGCT[r5]CGCGCACCTAGAGGGATATCACTCAGCATAATTAA -5' 3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNN -5' After fill in with taq polymerase (2X) (could use BST, T4 DNA poly also) 5'- [AATTAATACGACTCACTATAGGGAGA]TCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNN -3' 3'- [TTAATTATGCTGAGTGATATCCCTCT]AGGTGCGCG[r5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC (NNNNNNNNN)NNNN -5' 5'- NNNN(NNNNNNNNN) CTGTCTCTTATACACATCTGACGCTGCCGACGA[r5]GCGCGTGGA[TCTCCCTATAGTGAGTCGTATTAATT] -3' 3'- NNNNNNNNNNNNNNN GACAGAGAATATGTGTAGACTGCGACGGCTGCT[r5]CGCGCACCT[AGAGGGATATCACTCAGCATAATTAA] -5' After IVT, All RNA strands will have same 5' end. RNA is also purified after IVT. First G in GGG is the +1 site for the beginning of RNA synthesis for T7. for Tm's used used https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator (NEB Tm calculator) All Tm's calculated using, "taq DNA polymerase", "taq 2X Mastermix" RNA 5'- GGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNN -3' After single strand cDNA synthesis to form cDNA/RNA hybrid RNA 5'- GGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNN -3' cDNA 3'- CCCTCTAGGTGCGCG[r5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNN -5' After Rnase H nicking, denatuation of small RNAs? and addition of 5' end primer. Extension with BST or klenow exo- or DNAP1 or Taq2x (been using taq2x) DNA 5'- GGGAGATCCACGCGC -3' (Tm=57C, anneal at 52C) (sss_scnXTv2) cDNA 3'- CCCTCTAGGTGCGCG[r5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNN -5' Now have double stranded DNA, which should be long. DNA 5'- GGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNN -3' cDNA 3'- CCCTCTAGGTGCGCG[r5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNN -5'
Custom Nextera i7 transposome i7 adapter 5’- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3' (nXTv2_i7_top) 3'- TCTACACATATTCTCTGTC/5Phos/ -5' (P-nXTv2_i7_bot) 5'- /5Phos/CTGTCTCTTATACACATCT -3' (P-nXTv2_i7_bot) 3’- GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' (nXTv2_i7_top) After custom nextera tagmentation: DNA 5'- GGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3' cDNA 3'- CCCTCTAGGTGCGCG[r5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' After fill in: DNA 5'- GGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3' cDNA 3'- CCCTCTAGGTGCGCG[r5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' Amplification (to add i5/i7 barcodes): for Tm's used used https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator (NEB Tm calculator) 'All Tm's calculated using, "taq DNA polymerase", "taq 2X Mastermix" 5'- AATGATACGGCGACCACCGA -3' (i5_PCR_ILAdptr, Tm=59C) 5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCCACGCGC -3' (scT7_S5XX, comp region Tm=57C, whole~75C) DNA 5'- GGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3' cDNA 3'- CCCTCTAGGTGCGCG[r5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' (i7XX_orig, comp region Tm=57C, whole~71C) 3'- GGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5' (i7XX_long, comp region Tm=59C, whole~71C) 3'- GAGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5' (i7_PCR_ILAdptr, Tm=58C) 3'- AGCATACGGCAGAAGACGAAC -5' Final sequencing ready fragment nXTv2_i5_index_read -----> nXTv2_read1 -----> nXTv2_i7_index_read -----> 5'- AATGATACGGCGACCACCGAGATCTACAC -3' 5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3' 5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3' 5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC[i7]ATCTCGTATGCCGTCTTCTGCTTG -3' 3'- TTACTATGCCGCTGGTGGCTCTAGATGTG[i5]CCCTCTAGGTGCGCG[r5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5' 3'- AGCAGCCGTCGCAGTCTACACATATTCTCTGTC -5' 3'- GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' <----- r5_index_read <----- nXTv2_read2
Sequences to order scT7_r5XXX_i5_top Barcoded Transposons P-T7tspn-bot 5'- /5Phos/CTGTCTCTTATACACATCT -3' sss_scnXTv2 5'- GGGAGATCCACGCGC -3' nXTv2_i7_top 5’- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3' P-nXTv2_i7_bot 5'- /5Phos/CTGTCTCTTATACACATCT -3' scT7_S5XX Modified Nextera XT V2 i5 barcode sequences and adaptors i7XX_long Extended Nextera XT V2 i7 barcode sequences and adaptors i5_PCR_ILAdptr 5'- AATGATACGGCGACCACCGA -3' i7_PCR_ILAdptr 5'- CAAGCAGAAGACGGCATACGA-3' nXTv2_read1 5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3' nXTv2_i7_index_read 5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3' nXTv2_i5_index_read 5'- AATGATACGGCGACCACCGAGATCTACAC -3' r5_index_read 5'- CTGTCTCTTATACACATCTGACGCTGCCGACGA -3' nXTv2_read2 5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3'
Barcoded scT7_r5XXX_i5_top transposons 74 bp long, using truncated barcodes from Quantitative single-cell RNA-seq with unique molecular identifiers num. r5_barcode r5_smpl_sheet name r5_full_sequence_(5'_->_3') 1 TCTAAT ATTAGA scT7_r5001_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTCTAATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 2 ACTCGT ACGAGT scT7_r5002_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACTCGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 3 ACGTCA TGACGT scT7_r5003_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACGTCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 4 AACGAC GTCGTT scT7_r5004_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAACGACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 5 TTAGCG CGCTAA scT7_r5005_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTTAGCGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 6 ACAGAC GTCTGT scT7_r5006_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACAGACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 7 CGTAGG CCTACG scT7_r5007_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCGTAGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 8 GAGCTC GAGCTC scT7_r5008_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGAGCTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 9 AATACC GGTATT scT7_r5009_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAATACCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 10 TCACAC GTGTGA scT7_r5010_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTCACACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 11 CTGGTA TACCAG scT7_r5011_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCTGGTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 12 GATAGG CCTATC scT7_r5012_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGATAGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 13 TGTCGA TCGACA scT7_r5013_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTGTCGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 14 ACGGGT ACCCGT scT7_r5014_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACGGGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 15 CAGCAT ATGCTG scT7_r5015_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCAGCATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 16 TTGACA TGTCAA scT7_r5016_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTTGACATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 17 TTCAAG CTTGAA scT7_r5017_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTTCAAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 18 GTCCTG CAGGAC scT7_r5018_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGTCCTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 19 CACTGA TCAGTG scT7_r5019_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCACTGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 20 TACAGG CCTGTA scT7_r5020_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTACAGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 21 ACCTTT AAAGGT scT7_r5021_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACCTTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 22 CCGTTC GAACGG scT7_r5022_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCCGTTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 23 TGTTTG CAAACA scT7_r5023_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTGTTTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 24 GTGGCT AGCCAC scT7_r5024_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGTGGCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 25 GTACAA TTGTAC scT7_r5025_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGTACAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 26 CAAAGT ACTTTG scT7_r5026_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCAAAGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 27 GCATGA TCATGC scT7_r5027_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGCATGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 28 CATTAG CTAATG scT7_r5028_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCATTAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 29 CTCCTT AAGGAG scT7_r5029_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCTCCTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 30 ACCCGA TCGGGT scT7_r5030_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACCCGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 31 TGCCAC GTGGCA scT7_r5031_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTGCCACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 32 TACCAA TTGGTA scT7_r5032_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTACCAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 33 CGAGGT ACCTCG scT7_r5033_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCGAGGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 34 CCACCA TGGTGG scT7_r5034_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCCACCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 35 TAATCA TGATTA scT7_r5035_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTAATCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 36 AGTCCA TGGACT scT7_r5036_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAGTCCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 37 CTCAGC GCTGAG scT7_r5037_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCTCAGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 38 TGGACT AGTCCA scT7_r5038_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTGGACTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 39 CAAACC GGTTTG scT7_r5039_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCAAACCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 40 ACTCCG CGGAGT scT7_r5040_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACTCCGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 41 ACCGTG CACGGT scT7_r5041_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACCGTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 42 AGAACG CGTTCT scT7_r5042_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAGAACGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 43 ATTGAC GTCAAT scT7_r5043_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCATTGACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 44 GTATCC GGATAC scT7_r5044_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGTATCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 45 CGGAAA TTTCCG scT7_r5045_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCGGAAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 46 TTGTGT ACACAA scT7_r5046_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTTGTGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 47 GCACAT ATGTGC scT7_r5047_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGCACATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 48 GTCATT AATGAC scT7_r5048_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGTCATTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 49 CCTATA TATAGG scT7_r5049_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCCTATATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 50 ACGCTG CAGCGT scT7_r5050_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACGCTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 51 GATTGT ACAATC scT7_r5051_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGATTGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 52 ATGTAG CTACAT scT7_r5052_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCATGTAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 53 CGCTGT ACAGCG scT7_r5053_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCGCTGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 54 AGACTT AAGTCT scT7_r5054_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAGACTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 55 TGGGCA TGCCCA scT7_r5055_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTGGGCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 56 ACTAGA TCTAGT scT7_r5056_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACTAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 57 ATGGGA TCCCAT scT7_r5057_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCATGGGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 58 GCGACG CGTCGC scT7_r5058_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGCGACGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 59 GGGTAG CTACCC scT7_r5059_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGGGTAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 60 GGTGAA TTCACC scT7_r5060_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGGTGAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 61 CATCTT AAGATG scT7_r5061_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCATCTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 62 ATAATC GATTAT scT7_r5062_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCATAATCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 63 TATGGT ACCATA scT7_r5063_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTATGGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 64 GACTTA TAAGTC scT7_r5064_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGACTTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 65 TGATTC GAATCA scT7_r5065_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTGATTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 66 TAGCGA TCGCTA scT7_r5066_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTAGCGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 67 AACTCC GGAGTT scT7_r5067_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAACTCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 68 GAAGTG CACTTC scT7_r5068_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGAAGTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 69 GAGTAC GTACTC scT7_r5069_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGAGTACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 70 CGCAAT ATTGCG scT7_r5070_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCGCAATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 71 CTGGAG CTCCAG scT7_r5071_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCTGGAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 72 TGGGTT AACCCA scT7_r5072_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTGGGTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 73 TCCTAG CTAGGA scT7_r5073_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTCCTAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 74 TCATGC GCATGA scT7_r5074_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTCATGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 75 TTCCCT AGGGAA scT7_r5075_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTTCCCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 76 GTAAAG CTTTAC scT7_r5076_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGTAAAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 77 ACAACT AGTTGT scT7_r5077_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACAACTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 78 AGGCAT ATGCCT scT7_r5078_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAGGCATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 79 TGTGTC GACACA scT7_r5079_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTGTGTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 80 AGCATA TATGCT scT7_r5080_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAGCATATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 81 AGACGC GCGTCT scT7_r5081_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAGACGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 82 CTTGCT AGCAAG scT7_r5082_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCTTGCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 83 TCTGCC GGCAGA scT7_r5083_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTCTGCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 84 TAGTCG CGACTA scT7_r5084_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTAGTCGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 85 TTGCTG CAGCAA scT7_r5085_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTTGCTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 86 GACAAC GTTGTC scT7_r5086_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGACAACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 87 ATTCCC GGGAAT scT7_r5087_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCATTCCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 88 TTAAGC GCTTAA scT7_r5088_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTTAAGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 89 CGCCTC GAGGCG scT7_r5089_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCGCCTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 90 CTTATC GATAAG scT7_r5090_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCTTATCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 91 CGCTAA TTAGCG scT7_r5091_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCGCTAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 92 AGGTCC GGACCT scT7_r5092_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAGGTCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 93 AAGGTG CACCTT scT7_r5093_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAAGGTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 94 GTTGTA TACAAC scT7_r5094_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGTTGTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 95 AGAGAA TTCTCT scT7_r5095_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAGAGAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 96 ATTTGG CCAAAT scT7_r5096_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCATTTGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
Modified Nextera XT V2 i5 barcode sequences and adaptors same i7 adaptors/barcodes are used as in original Nextera XT V2 system or 2 bp longer ones can be used as listed below num. i5_barcode i5_smplsheet scT7_i5_name scT7_i5_full_sequence_(5'_->_3') 1 CTCTCTAT CTCTCTAT scT7_S502 AATGATACGGCGACCACCGAGATCTACACCTCTCTATGGGAGATCCACGCGC 2 TATCCTCT TATCCTCT scT7_S503 AATGATACGGCGACCACCGAGATCTACACTATCCTCTGGGAGATCCACGCGC 3 GTAAGGAG GTAAGGAG scT7_S505 AATGATACGGCGACCACCGAGATCTACACGTAAGGAGGGGAGATCCACGCGC 4 ACTGCATA ACTGCATA scT7_S506 AATGATACGGCGACCACCGAGATCTACACACTGCATAGGGAGATCCACGCGC 5 AAGGAGTA AAGGAGTA scT7_S507 AATGATACGGCGACCACCGAGATCTACACAAGGAGTAGGGAGATCCACGCGC 6 CTAAGCCT CTAAGCCT scT7_S508 AATGATACGGCGACCACCGAGATCTACACCTAAGCCTGGGAGATCCACGCGC 7 CGTCTAAT CGTCTAAT scT7_S510 AATGATACGGCGACCACCGAGATCTACACCGTCTAATGGGAGATCCACGCGC 8 TCTCTCCG TCTCTCCG scT7_S511 AATGATACGGCGACCACCGAGATCTACACTCTCTCCGGGGAGATCCACGCGC 9 TCGACTAG TCGACTAG scT7_S513 AATGATACGGCGACCACCGAGATCTACACTCGACTAGGGGAGATCCACGCGC 10 TTCTAGCT TTCTAGCT scT7_S515 AATGATACGGCGACCACCGAGATCTACACTTCTAGCTGGGAGATCCACGCGC 11 CCTAGAGT CCTAGAGT scT7_S516 AATGATACGGCGACCACCGAGATCTACACCCTAGAGTGGGAGATCCACGCGC 12 GCGTAAGA GCGTAAGA scT7_S517 AATGATACGGCGACCACCGAGATCTACACGCGTAAGAGGGAGATCCACGCGC 13 CTATTAAG CTATTAAG scT7_S518 AATGATACGGCGACCACCGAGATCTACACCTATTAAGGGGAGATCCACGCGC 14 AAGGCTAT AAGGCTAT scT7_S520 AATGATACGGCGACCACCGAGATCTACACAAGGCTATGGGAGATCCACGCGC 15 GAGCCTTA GAGCCTTA scT7_S521 AATGATACGGCGACCACCGAGATCTACACGAGCCTTAGGGAGATCCACGCGC 16 TTATGCGA TTATGCGA scT7_S522 AATGATACGGCGACCACCGAGATCTACACTTATGCGAGGGAGATCCACGCGC num. i7_barcode i7_name i7_smplsheet i7_full_sequence_(5'_->_3') 1 TCGCCTTA N701_long TAAGGCGA CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGGAG 2 CTAGTACG N702_long CGTACTAG CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGGAG 3 TTCTGCCT N703_long AGGCAGAA CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGGAG 4 GCTCAGGA N704_long TCCTGAGC CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCGTGGGCTCGGAG 5 AGGAGTCC N705_long GGACTCCT CAAGCAGAAGACGGCATACGAGATAGGAGTCCGTCTCGTGGGCTCGGAG 6 CATGCCTA N706_long TAGGCATG CAAGCAGAAGACGGCATACGAGATCATGCCTAGTCTCGTGGGCTCGGAG 7 GTAGAGAG N707_long CTCTCTAC CAAGCAGAAGACGGCATACGAGATGTAGAGAGGTCTCGTGGGCTCGGAG 8 CAGCCTCG N710_long CGAGGCTG CAAGCAGAAGACGGCATACGAGATCAGCCTCGGTCTCGTGGGCTCGGAG 9 TGCCTCTT N711_long AAGAGGCA CAAGCAGAAGACGGCATACGAGATTGCCTCTTGTCTCGTGGGCTCGGAG 10 TCCTCTAC N712_long GTAGAGGA CAAGCAGAAGACGGCATACGAGATTCCTCTACGTCTCGTGGGCTCGGAG 11 TCATGAGC N714_long GCTCATGA CAAGCAGAAGACGGCATACGAGATTCATGAGCGTCTCGTGGGCTCGGAG 12 CCTGAGAT N715_long ATCTCAGG CAAGCAGAAGACGGCATACGAGATCCTGAGATGTCTCGTGGGCTCGGAG 13 TAGCGAGT N716_long ACTCGCTA CAAGCAGAAGACGGCATACGAGATTAGCGAGTGTCTCGTGGGCTCGGAG 14 GTAGCTCC N718_long GGAGCTAC CAAGCAGAAGACGGCATACGAGATGTAGCTCCGTCTCGTGGGCTCGGAG 15 TACTACGC N719_long GCGTAGTA CAAGCAGAAGACGGCATACGAGATTACTACGCGTCTCGTGGGCTCGGAG 16 AGGCTCCG N720_long CGGAGCCT CAAGCAGAAGACGGCATACGAGATAGGCTCCGGTCTCGTGGGCTCGGAG 17 GCAGCGTA N721_long TACGCTGC CAAGCAGAAGACGGCATACGAGATGCAGCGTAGTCTCGTGGGCTCGGAG 18 CTGCGCAT N722_long ATGCGCAG CAAGCAGAAGACGGCATACGAGATCTGCGCATGTCTCGTGGGCTCGGAG 19 GAGCGCTA N723_long TAGCGCTC CAAGCAGAAGACGGCATACGAGATGAGCGCTAGTCTCGTGGGCTCGGAG 20 CGCTCAGT N724_long ACTGAGCG CAAGCAGAAGACGGCATACGAGATCGCTCAGTGTCTCGTGGGCTCGGAG 21 GTCTTAGG N726_long CCTAAGAC CAAGCAGAAGACGGCATACGAGATGTCTTAGGGTCTCGTGGGCTCGGAG 22 ACTGATCG N727_long CGATCAGT CAAGCAGAAGACGGCATACGAGATACTGATCGGTCTCGTGGGCTCGGAG 23 TAGCTGCA N728_long TGCAGCTA CAAGCAGAAGACGGCATACGAGATTAGCTGCAGTCTCGTGGGCTCGGAG 24 GACGTCGA N729_long TCGACGTC CAAGCAGAAGACGGCATACGAGATGACGTCGAGTCTCGTGGGCTCGGAG
original Nextera XT V2 i5/i7 barcode sequences and primers num. i5_barcode i5_name i5_smplsheet i5_full_sequence_(5'_->_3') 1 CTCTCTAT S502 CTCTCTAT AATGATACGGCGACCACCGAGATCTACACCTCTCTATTCGTCGGCAGCGTC 2 TATCCTCT S503 TATCCTCT AATGATACGGCGACCACCGAGATCTACACTATCCTCTTCGTCGGCAGCGTC 3 GTAAGGAG S505 GTAAGGAG AATGATACGGCGACCACCGAGATCTACACGTAAGGAGTCGTCGGCAGCGTC 4 ACTGCATA S506 ACTGCATA AATGATACGGCGACCACCGAGATCTACACACTGCATATCGTCGGCAGCGTC 5 AAGGAGTA S507 AAGGAGTA AATGATACGGCGACCACCGAGATCTACACAAGGAGTATCGTCGGCAGCGTC 6 CTAAGCCT S508 CTAAGCCT AATGATACGGCGACCACCGAGATCTACACCTAAGCCTTCGTCGGCAGCGTC 7 CGTCTAAT S510 CGTCTAAT AATGATACGGCGACCACCGAGATCTACACCGTCTAATTCGTCGGCAGCGTC 8 TCTCTCCG S511 TCTCTCCG AATGATACGGCGACCACCGAGATCTACACTCTCTCCGTCGTCGGCAGCGTC 9 TCGACTAG S513 TCGACTAG AATGATACGGCGACCACCGAGATCTACACTCGACTAGTCGTCGGCAGCGTC 10 TTCTAGCT S515 TTCTAGCT AATGATACGGCGACCACCGAGATCTACACTTCTAGCTTCGTCGGCAGCGTC 11 CCTAGAGT S516 CCTAGAGT AATGATACGGCGACCACCGAGATCTACACCCTAGAGTTCGTCGGCAGCGTC 12 GCGTAAGA S517 GCGTAAGA AATGATACGGCGACCACCGAGATCTACACGCGTAAGATCGTCGGCAGCGTC 13 CTATTAAG S518 CTATTAAG AATGATACGGCGACCACCGAGATCTACACCTATTAAGTCGTCGGCAGCGTC 14 AAGGCTAT S520 AAGGCTAT AATGATACGGCGACCACCGAGATCTACACAAGGCTATTCGTCGGCAGCGTC 15 GAGCCTTA S521 GAGCCTTA AATGATACGGCGACCACCGAGATCTACACGAGCCTTATCGTCGGCAGCGTC 16 TTATGCGA S522 TTATGCGA AATGATACGGCGACCACCGAGATCTACACTTATGCGATCGTCGGCAGCGTC num. i7_barcode i7_name i7_smplsheet i7_full_sequence_(5'_->_3') 1 TCGCCTTA N701 TAAGGCGA CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGG 2 CTAGTACG N702 CGTACTAG CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGG 3 TTCTGCCT N703 AGGCAGAA CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGG 4 GCTCAGGA N704 TCCTGAGC CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCGTGGGCTCGG 5 AGGAGTCC N705 GGACTCCT CAAGCAGAAGACGGCATACGAGATAGGAGTCCGTCTCGTGGGCTCGG 6 CATGCCTA N706 TAGGCATG CAAGCAGAAGACGGCATACGAGATCATGCCTAGTCTCGTGGGCTCGG 7 GTAGAGAG N707 CTCTCTAC CAAGCAGAAGACGGCATACGAGATGTAGAGAGGTCTCGTGGGCTCGG 8 CAGCCTCG N710 CGAGGCTG CAAGCAGAAGACGGCATACGAGATCAGCCTCGGTCTCGTGGGCTCGG 9 TGCCTCTT N711 AAGAGGCA CAAGCAGAAGACGGCATACGAGATTGCCTCTTGTCTCGTGGGCTCGG 10 TCCTCTAC N712 GTAGAGGA CAAGCAGAAGACGGCATACGAGATTCCTCTACGTCTCGTGGGCTCGG 11 TCATGAGC N714 GCTCATGA CAAGCAGAAGACGGCATACGAGATTCATGAGCGTCTCGTGGGCTCGG 12 CCTGAGAT N715 ATCTCAGG CAAGCAGAAGACGGCATACGAGATCCTGAGATGTCTCGTGGGCTCGG 13 TAGCGAGT N716 ACTCGCTA CAAGCAGAAGACGGCATACGAGATTAGCGAGTGTCTCGTGGGCTCGG 14 GTAGCTCC N718 GGAGCTAC CAAGCAGAAGACGGCATACGAGATGTAGCTCCGTCTCGTGGGCTCGG 15 TACTACGC N719 GCGTAGTA CAAGCAGAAGACGGCATACGAGATTACTACGCGTCTCGTGGGCTCGG 16 AGGCTCCG N720 CGGAGCCT CAAGCAGAAGACGGCATACGAGATAGGCTCCGGTCTCGTGGGCTCGG 17 GCAGCGTA N721 TACGCTGC CAAGCAGAAGACGGCATACGAGATGCAGCGTAGTCTCGTGGGCTCGG 18 CTGCGCAT N722 ATGCGCAG CAAGCAGAAGACGGCATACGAGATCTGCGCATGTCTCGTGGGCTCGG 19 GAGCGCTA N723 TAGCGCTC CAAGCAGAAGACGGCATACGAGATGAGCGCTAGTCTCGTGGGCTCGG 20 CGCTCAGT N724 ACTGAGCG CAAGCAGAAGACGGCATACGAGATCGCTCAGTGTCTCGTGGGCTCGG 21 GTCTTAGG N726 CCTAAGAC CAAGCAGAAGACGGCATACGAGATGTCTTAGGGTCTCGTGGGCTCGG 22 ACTGATCG N727 CGATCAGT CAAGCAGAAGACGGCATACGAGATACTGATCGGTCTCGTGGGCTCGG 23 TAGCTGCA N728 TGCAGCTA CAAGCAGAAGACGGCATACGAGATTAGCTGCAGTCTCGTGGGCTCGG 24 GACGTCGA N729 TCGACGTC CAAGCAGAAGACGGCATACGAGATGACGTCGAGTCTCGTGGGCTCGG
Validation of THS-seq barcoded transposons using Nextera XT v2 sequences for multiplex combinatorial indexing
- designed scT7_r5XXX_i5_top transposon so it can be used used with 6 base pair barcodes. 96 barcoded transposons made. Will first test with tn5059 transposomes from Trina for validation the transposon is working and that fragments can be sequenced on MiSeq/HiSeq. Then will proceed to order the rest of the transposons and use with combinatorial sequencing with THS-seq for single cells.
- There is slight modification to MiSeq protocol, custom primers will need to be used and Read2 will instead be a 6 basepair index reading using r5_index_read. Standard Nextera XT v2 oligos i5 index, i7 index and read1 are used for sequencing.
Before starting protocols
1. Check if have enough reagents etc for the protocol
- lysis buffer
- tn5059 transposomes
- custom 3' transpson
- IVT reagents
- cells etc
- 5X, 2X taq polymerase
- Zymo DNA clean and concentrator
- 2. Samples
samples 1. scT7_i502-i701, scT7_r5001_i5_top, 5X tn5-059, 500 cells rep1 2. scT7_i503-i701, scT7_r5001_i5_top, 5X tn5-059, 500 cells rep2 3. scT7_i505-i701, scT7_r5002_i5_top, 5X tn5-059, 500 cells rep1 4. scT7_i502-i702, scT7_r5002_i5_top, 5X tn5-059, 500 cells rep2 5. scT7_i503-i702, scT7_r5001_i5_top, 5X tn5-059, 6 ng pure DNA 6. scT7_i505-i702, scT7_r5001_i5_top, 5X tn5-059, NTC
IVT Protocol
- If need to make more transposome, do first 2 steps. If not goto step 3.
Generation of transposomes
1. annealing of ME sequence to T7 transposon sequence
a. Make 100 uM stock solution of tranposon and ME only bottom end. b. Incubate 15 uL of each oligo (100uM) with 20 uL EB buffer at 95C for 2 minutes, then cool to 14C at 0.1 C/s. c. Oligo's now at 30 uM in 50 uL.
2. Transposome complex generation, run controls!!!
- add the below components into one tube and incubate for 30 minutes at RT
a. Add 1.0 uL of 100% sterile glycerol to tube b. Add 1.0 uL of annealed transposon to the 100% sterile glycerol and mix well c. Add 2.0 uL of transposase to well.
- store at -20, is good for a year
3. Prepare samples, lyse cells with lysis buffer
CELL WASHING - (wash cells with PBS)
a. count cells, spin down all cells at 250Xg for 5 minutes b. wash with 1X PBS, centrifuge 250Xg for 5 minutes, resuspend to 1000 cells/uL c. Count cells again to be sure of cells/uL amount.
CELL LYSIS
a. dilute Lysis Buffer (LB) to 2X, or as needed (from 10X stock) b. add 1 uL LB to sample tubes c. add 1 uL cells to sample tubes with LB in it, mix briefly (mineral oil optional) d. transposition ready
LYSIS BUFFER NOTES
- Lysis buffer is: 10ml 10X lysis buffer (LB, 100mM Tris.Hcl pH 7.5, 100mM NaCl, 30mM MgCl2, 1% NP40, Crawford et al. PNAS 2003) in nuclease free H2O.
4. transposition reaction. Add all components and incubate at 37C FOR 30 MINUTES
1.0 uL 5X Custom Tagmentation buffer 2.0 uL lysed cells/pure genomic DNA 1.0 uL T7 transposomes (5X tn5-059, indexed Tsome as listed) 1.0 uL N-H2O ___________ 5.0 uL total solution
5. Bring to 15 uL with N-H2O. Add 15 uL 8M guanidine HCl for a total of 30 uL.
- For SPRI beads add 1.8X (54 uL) beads per sample. and follow beads protocol
- ELUTE IN 9.6, leave beads in solution
6. Fill in reaction
- Add 2.4 uL of 5X taq polymerase, mix thoroughly. Run at 72C for 3 minutes.
7. Maxiscript (Ambion) T7 Protocol, IVT
- DNA from PCR can be used directly in the MAXIscript Kit without any pretreatment or purification.
a. Thaw 10X Transcription Buffer and ribonucleotide solutions. Store the ribonucleotides (A, C, G, U) on ice, but keep 10X transcription buffer at room temp b. Assemble reaction mixture at room temperature, ADD IN ORDER AND MIX THOROUGHLY!!!! X uL DNA template (list 1 ug) 2 uL 10X Transcription Buffer 1 uL 10 mM ATP 1 uL 10 mM CTP 1 uL 10 mM GTP 1 uL 10 mM UTP 2 uL T7 Enzyme Mix _______________________ 20 uL total b. Incubate reactions at 37C overnight for ~16-19 hours. (>10 uM limiting nucleotide)
8. Clean with Zymo RNA clean and concentrator, put solution and all beads on column.
- elute samples in 10 uL of N-H2O
- quanitate/view on TBU gel.
AFTER IVT RNA HAS ALREADY BEEN GENERATED AND CLEANED WITH ZYMO
sample list 1. dkljd 2. ldkf
- Amounts of RNA and water to add for MMLV RT (50 ng RNA)
9. single strand synthesis MMLV RT (Clontech)
- Followed protocol for SMART MMLV Reverse Transcriptase
a. Add 2.5 uL 20 uM (100 ng total) RANDOM HEXAMERS to RNA sample. Bring to final volume of 11.5 uL with Nuclease free H2O b. heat the mixture to 70C for 3 minutes. Immediately cool on ice. c. Add the following to the reaction. 4 uL 5X first strand buffer 2 uL dNTP mix 2 uL 100 mM DTT .5 uL SMART MMLV RT and mix (ADD LAST!!!!!) ___________________________ 20 uL total d. Incuvate first at Room Temperature for 10 minutes. Then incubate at 42C for 60 minutes. e. Terminate the reaction by heating at 70C for 10 minutes
10. RNase H digestion
- Use .5 Units for 20 uL reaction. Stock is at 5 Units/ul, thus dilute 10X and use 1 uL per reaction.
a. Add 1 uL of 10X diluted Rnase H to the reaction.
b. Incubate for 20 minutes at 37C.
11. Second strand synthesis: Adding sss_scnXTv2 primer and polymerases. Can incubate at higher temps (usually at 16C since RNA is nicked) since sss_scnXTv2 has a high Tm.
a. Add 2.5 uL of 20 uM sss_scnXTv2 to each reaction. b. Incubate solutions for 2 minutes at 65C. Cool immediately on ice. c. Add 5.875 uL taq5X to reactions, Incubate at 72C for 8 minutes
12. Clean DNA with AMPURE beads depending. elute in 20 uL and concentrate.
- USED AMPURE DNA BEADS AT 1:1.8X RATIO
- add 45 uL N-H2O
- add 135 uL beads per reaction
- elute on 20 uL
13. After elution in 20 uL, use vacuum centrifuge commentator to concentrate sample size to ~4-5 uL per sample.
- Also ~4-5 uL per sample is preferred for transposition, which has always been performed in low volume solutions.
RAN FOR 16 MINUTES
14. Fragmenting and 3' End tagging with [1X] custom nXTv2_i7 Tn5059 transposome and custom tagmentation buffer D6
1 uL custom tagmentation buffer X uL cDNA sample X uL Nuclase free H2O 1 uL prepared 1X T7 transposomes (MAKE SURE TO ADD LAST) ___________ 5 uL total solution Incubate at 55C for 6 minutes, cool briefly on ice after
15. Protease digestion of transposase, protease inactivation
To each tube, add: 1 uL Qiagen Protease, for 5 uL reaction 1 uL of .5 for .1 AU final. (stock is 5 AU and diluted 10X. want .5 AU/uL final []) Incubate: 50C 10 minutes, 70C 20 minutes
16. Fill in reaction
- Add 6 uL 2X taq polymerase, run at 72C for 3 minutes.
17. PCR addition of barcodes USING NEXTERA XT V2 modified i5 (scT7_S5XX) AND i7 (N7XX) SEQUENCES!!!!!
Samples: 1. dlkd 2. fdko
KAPA SYBR FAST qPCR mix until saturation, X35 cycles 9 uL Taq2X 2 uL scT7_S5XX index primer, 2 uL F, (scT7_S5XX) USED XXX 2 uL nXTv2_i7 index primer, 2 uL R, (i7XX_long or i7XX_orig) 4 uL H2O 1 uL 25X SYBR green 12 uL DNA template (use half RT reaction) _____________ 30 uL KAPA SYBR cycles: 72C 3m, 95C 30s, (95C for 10s, 63C for 30s, 72C for 1:30 min) X15, 72C for 3 min, 4C forever
- terminate before curves saturate (usually cycle 6-8)
18. Gel Size selection
- gel size select from 220-1000 bp, follow gel size selection protocol
- do not need to include controls.
Results
- total RNA.
- TBU gel after IVT, Guanidine HCl to remove proteins
- qPCR curves
- after adding barcodes, TBE gel
- gel size selection amounts
- gel size selection
- gel size selection validation
conclusions
- submitted for sequencing